The long-term goal of the project is to investigate the role of p53 in osteoblast differentiation, a process central to bone formation. In our studies with osteosarcomas, we have shown that function al loss of p53 predominates, and replacing the wild type p53 gene in to these cells not only results in suppression of growth, but in the induction of osteoblast differentiation. We have identified a bone specific, differentiation related gene, osteocalcin, to be regulated by p53. While a role for p53 in tissue differentiation is increasingly being accepted, it has been difficult to define. We propose that p53 provides the right internal environment for differentiation by factoring the in put from several sources, due to its involvement in cellular pathways like apoptosis and cell cycle arrest. We have found p53 activity to be selectively maintained late during the differentiation process, coinciding with the upregulation of bone specific gene expression. The proposed experiments are therefore designed to understand: a). The nature of p53-dependent gene expression during osteoblast differentiation. b). The type of regulation that allows the maintenance of p53 expression. c). Some of the bone specific targets of p53's transcription activation. The focus of the research will be: a). To identify the p53-regulated genes that are modulated during differentiation. b). To determine how the mdm2 and p19ARF genes affect p53 activity during osteoblast differentiation. c). On how p53 directly regulates the activity of the bone specific, differentiation related osteocalcin gene, and bone morphogenetic protein 2 (BMP-2). We anticipate that the proposed studies will generate valuable new data, shed more light on the physiological roles of the above proteins, and advance our knowledge of the mechanisms responsible for osteoblast cell differentiation. A better understanding of this basic aspect of cell biology could also potentially lead to additional benefits in diseases involving bone and cancer. Elucidation of the mechanisms whereby tumors block cell differentiation could make possible the development of strategies to circumvent them. The information generated would enhance our understanding of p53 and provide further avenues for gene therapy modalities to be extended to patients with osteosarcomas and other bone disorders such as rheumatoid arthritis, where p53 mutations predominate.